The majority of villas are on the Istria peninsula, with others across Split, Dubrovnik and the Dalmatian islands such as Brac and Hvar.
Prices for a seven-night booking lead in from £279 in low season, while a family villa with a private pool in August starts from £829.
The new Croatia properties have been added to James Villas’ inventory from sister brand Novasol.
James Villas said it had “consolidated its growth plans” during the past two years with a focus on growing its choice of accommodation-only holidays in Europe – now claiming to offer more than 7,000 properties.
Commercial director Dave Woollcott said the company had seen “significant demand for more choice of accommodation” in Croatia.